Fig. 3. Immunolocalization of endogenous hARL5 in hepatoma Hep3B and Huh7 cells. (A) Specificity of antibody against ARL5. 100 ng of the indicated purified recombinant His-tagged ARL (a,b) and the indicated amounts of purified His-tagged hARL5 (c) were subjected to SDS-PAGE in 12.5% gels. Positions of protein standards (32.9 and 24.8 kDa) are on the left. Proteins were transferred to nitrocellulose and reacted with (a) anti-Histag or (b,c) anti-ARL5 antibodies, followed by detection using the ECL system (A,B). (B) Subcellular distribution of hARL5. Nuclear (N), membrane (M) and cytosolic (C) fractions of Hepatoma Hep3B or Huh7 cells were prepared as described in Materials and Methods. Equivalent amounts (from total homogenate) of each fraction were analyzed by electrophoresis and immunoblotting using specific antibodies against ARL5, -tubulin (cytosolic marker), or PCNA (nuclear marker). (C) Immunolocalization of ARL5. Hepatoma Hep3B and Huh7 cells on glass coverslips, treated as described in Materials and Methods, were incubated with affinity-purified anti-hARL5-peptide (a,d) or ßCOP antibodies (c,f). Coverslips were mounted on Mowiol (supplemented with Hoechst 33258; b,e) and inspected with a Zeiss Axiophot equipped for epifluorescence. Bar, 10 µm.